Immunohistochemical Evaluation of Adaptor Protein FAM159B Expression in Normal and Neoplastic Human Tissues

FAM159B is a so-called adaptor protein. These proteins are essential components in numerous cell signalling pathways. However, little is known regarding FAM159B expression in normal and neoplastic human tissues. The commercially available rabbit polyclonal anti-human FAM159B antibody HPA011778 was initially characterised for its specificity using Western blot analyses and immunocytochemistry and then applied to a large series of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. Confirmation of FAM159B’s predicted size and antibody specificity was achieved in BON-1 cells, a neuroendocrine tumour cell line endogenously expressing FAM159B, using targeted siRNA. Immunocytochemical experiments additionally revealed cytoplasmic expression of the adaptor protein. Immunohistochemical staining detected FAM159B expression in neuronal and neuroendocrine tissues such as the cortex, the trigeminal ganglia, dorsal root and intestinal ganglia, the pancreatic islets and the neuroendocrine cells of the bronchopulmonary and gastrointestinal tract, but also in the syncytiotrophoblasts of the placenta. FAM159B was also expressed in many of the 28 tumour entities investigated, with high levels in medullary and anaplastic thyroid carcinomas, parathyroid adenomas, lung and ovarian carcinomas, lymphomas and neuroendocrine tumours of different origins. The antibody HPA011778 can act as a useful tool for basic research and identifying FAM159B expression in tissue samples.


Introduction
Adaptor proteins are usually non-enzymes and are essential components that inherit important roles in various cell signalling pathways. In order to mediate protein-protein or protein-membrane interactions, they contain modular domains and/or linear peptide motifs. They are involved in regulating diverse aspects of cell surface receptor functions, including downstream signal transduction and transport or recycling of receptors. β-arrestin and its involvement in G-protein-coupled receptor signalling is a notable example of such an adaptor protein and its importance in the cell [1].
Another adaptor protein that is a Shisa-like protein is FAM159B. These Shisa-like proteins possess an N-terminal domain with six conserved cysteines and are thus related to the Shisa family of single transmembrane proteins that contain eight conserved cysteines. Vertebrates possess two forms of FAM159: FAM159A and FAM159B. FAM159B is thought to be a transmembrane adaptor that regulates other transmembrane proteins and receptors [1].
To identify genes with elevated expression in the human pancreas, Danielsson et al. [2] performed a genome-wide RNA sequencing analysis of the human transcriptome, followed by immunohistochemistry-based protein profiling to map proteins to their corresponding compartment(s) and cell type(s) within the pancreas. Among the identified upregulated proteins, several had not previously been described, including FAM159B, which was highly and specifically expressed in the islets and located at the cell membrane and in the cytoplasm. Distinct expression in neuroendocrine cells of the stomach mucosa was also observed, and FAM159B expression was consistent across all islet samples analysed from patients with type 1 or type 2 diabetes, as well as non-diabetic patients. The whole of the Langerhans islets showed FAM159B expression, leading to the conclusion that staining was probably not β-cell-specific. Therefore, FAM159B was proposed as a potential marker for total islet mass [2].
Using a simultaneous pancreatic single-cell, patch-clamp and RNA sequencing (patchseq) approach to identify genes associated with islet cell function and dysfunction, Camunas-Soler et al. [3] found that FAM159B transcripts correlated positively with total exocytosis, early exocytosis, Ca 2+ entry and Na + currents of β-cells. Furthermore, after knockdown of FAM159B with a specific siRNA, a significant reduction of the exocytotic response was observed. In contrast to the findings of Danielsson et al. [2], FAM159B was thus proposed to be a β-cell-enriched molecule, a regulator of β-cell exocytosis and a marker of β-cell subsets. In another study, single-cell transcriptome profiling of human stem-cell-derived islet cells before and 6 months after transplantation into the kidney capsule of diabetic mice was performed, and the results were compared to data obtained from native cadaveric human islets [4]. Whereas FAM159B was expressed in native islets, its expression was not detectable in non-grafted β-cells, although distinct upregulation of its transcripts upon maturation after grafting was noted. These findings were further confirmed by immunohistochemical stains of in vitro-derived and transplanted stem cells, indicating FAM159B as a possible maturation marker of β-cells.
Beyond these limited data regarding the presence of FAM159B in adult islet cells and endocrine cells of the gut, little is known of FAM159B expression in other normal human tissues. Notably, no information is available related to the possible presence of FAM159B in human tumours and cancer cell lines.
Therefore, the aim of the present study was to evaluate the expression of FAM159B in a large series of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples using immunohistochemistry to obtain a broad expression profile of this protein in humans and elucidate its possible functions. For this purpose, the commercially available rabbit polyclonal anti-human FAM159B antibody HPA011778 (Atlas Antibodies, Bromma, Sweden) was initially characterised for its specificity using Western blot analyses and immuncytochemistry in the neuroendocrine tumour cell line BON-1, which endogenously expresses FAM159B. Our subsequent immunohistochemical investigations revealed high expression of FAM159B, particularly in pancreas islets, specific neuroendocrine cells of the intestinal tract and small-cell lung cancer (SCLC). Based on these findings, the range of neuroendocrine neoplasms studied was broadened and, ultimately, a diverse panel of bronchopulmonary and gastroenteropancreatic neoplasms (BP-NEN and GEP-NEN) of different malignancy and origin were examined for FAM159B expression. Staining results were then correlated with clinical data, such as tumour stage and grade, glucagon or insulin secretion and overall survival of the patients, and with the expression data of different neuroendocrine tumour markers.

Characterisation of the Rabbit Polyclonal Anti-Human FAM159B Antibody HPA011778
The specificity of HPA011778 was first tested using Western blot analyses. When the cytosolic fraction of endogenously FAM159B-expressing BON-1 cells was separated through electrophoresis and immunoblotted, the antibody recognised a band at approximately M r = 16,000-18,000 ( Figure 1A).

Characterisation of the Rabbit Polyclonal Anti-Human FAM159B Antibody HPA011778
The specificity of HPA011778 was first tested using Western blot analyses. When the cytosolic fraction of endogenously FAM159B-expressing BON-1 cells was separated through electrophoresis and immunoblotted, the antibody recognised a band at approximately Mr = 16,000-18,000 ( Figure 1A). Western blot analysis of the supernatant from whole-cell preparations of BON-1 cells, which endogenously express FAM159B, after transfection with empty vector (scrambled) as a control (A) or specific siRNA (B). (C) Western blot analysis of wheat germ agarose (WGA) bead fractions containing glycosylated proteins. (D) Western blot analysis of the supernatant from whole-cell preparations when the antibody was pre-adsorbed with the immunising peptide. (E) Western Blot analysis of FAM159B in the cytosolic fraction of whole-organ preparations from mouse pancreas. Ordinate: migration of protein molecular weight markers (kDa). Representative results from one of three independent experiments are shown.
The observed molecular weight matched the expected weight of FAM159B, with Mr = 17,663 [5]. In contrast, no immunosignal could be detected in the wheat germ agarose (WGA) bead fraction, which contained the enriched glycosylated proteins ( Figure 1C). The observed molecular weight matched the expected weight of FAM159B, with M r = 17,663 [5]. In contrast, no immunosignal could be detected in the wheat germ agarose (WGA) bead fraction, which contained the enriched glycosylated proteins ( Figure 1C). After treatment of BON-1 cells with a targeted siRNA, the immunosignal in the cytosolic fraction was diminished ( Figure 1B). To further confirm the specificity of the antibody, it was preadsorbed with its immunising peptide and then used for Western blot analysis, which revealed that the immunosignal was completely abolished ( Figure 1D). As an endogenous control, immunoblots from the cytosolic fraction of whole-organ preparations from mouse pancreas were performed. Here, again, a band at approximately M r = 16,000-18,000 was detected ( Figure 1E).
For further evaluation of antibody specificity and characterisation of the subcellular localisation of the adaptor protein, we performed immunocytochemical analyses on BON-1 cells (Figure 2). The cells were left untreated or were transfected with a specific siRNA or an empty vector as control. They were then fixed and immunofluorescently stained with HPA011778. The untreated cells showed a bright immunosignal located throughout the cytosol. The signal remained the same with respect to its distribution pattern, but its intensity was reduced significantly in siRNA-transfected cells. BON-1 cells transfected as a control with an empty vector showed a similar immunosignal distribution and intensity to untreated cells. After pre-adsorption of the antibody with the peptide used for rabbit immunisations, complete repression of the immunosignal was again observed.
fraction was diminished ( Figure 1B). To further confirm the specificity of the antibody, it was pre-adsorbed with its immunising peptide and then used for Western blot analysis, which revealed that the immunosignal was completely abolished ( Figure 1D). As an endogenous control, immunoblots from the cytosolic fraction of whole-organ preparations from mouse pancreas were performed. Here, again, a band at approximately Mr = 16,000-18,000 was detected ( Figure 1E).
For further evaluation of antibody specificity and characterisation of the subcellular localisation of the adaptor protein, we performed immunocytochemical analyses on BON-1 cells (Figure 2). The cells were left untreated or were transfected with a specific siRNA or an empty vector as control. They were then fixed and immunofluorescently stained with HPA011778. The untreated cells showed a bright immunosignal located throughout the cytosol. The signal remained the same with respect to its distribution pattern, but its intensity was reduced significantly in siRNA-transfected cells. BON-1 cells transfected as a control with an empty vector showed a similar immunosignal distribution and intensity to untreated cells. After pre-adsorption of the antibody with the peptide used for rabbit immunisations, complete repression of the immunosignal was again observed.

Figure 2.
Immuncytochemical analysis of BON-1 cells endogenously expressing FAM159B. Cells remained untreated or were transfected with specific siRNA or an empty vector (scrambled) and then fixed and immunofluorescently stained with anti-FAM159B HPA011778. Right panel: staining using antibody HPA011778 pre-adsorbed with the immunising peptide. Green: immunosignal; blue: DAPI staining of DNA. All photomicrographs were captured at the same magnification. Scale bar: 100 µm.

Immunohistochemical Detection of FAM159B Expression in Normal Human Tissues
The rabbit polyclonal anti-FAM159B antibody HPA011778 was then applied to immunohistochemical stainings of different human non-neoplastic tissue samples. For most samples, immunostaining was localised to the cytoplasm of the cells, but in some cases a staining of the plasma membrane was also observed. A selection of FAM159B-positive normal and neoplastic human tissue samples was incubated with HPA011778 pre-adsorbed with its immunising peptide, which in all cases caused a complete loss of the immunosignal (see insets in Figures 3 and 7).

Immunohistochemical Detection of FAM159B Expression in Normal Human Tissues
The rabbit polyclonal anti-FAM159B antibody HPA011778 was then applied to immunohistochemical stainings of different human non-neoplastic tissue samples. For most samples, immunostaining was localised to the cytoplasm of the cells, but in some cases a staining of the plasma membrane was also observed. A selection of FAM159B-positive normal and neoplastic human tissue samples was incubated with HPA011778 pre-adsorbed with its immunising peptide, which in all cases caused a complete loss of the immunosignal (see insets in Figure 3 and Figure 7).
A distinct staining was detected in the pyramidal cells of the human cortex, trigeminal ganglia, dorsal root and intestinal ganglia, pancreatic islets, endocrine cells of the bronchopulmonary and intestinal tract and syncytiotrophoblasts of the placenta ( Figure 3). In addition, slight immunostaining was noticed in bronchial and intestinal epithelia, in the epithelium of the larger bile ducts in the liver and in that of the gallbladder. Within human kidneys, the adaptor protein was found in mesangial cells of the glomeruli, the visceral and parietal layer of Bowman's capsule and the distal tubules ( Figure 3E). No expression of FAM159B was observed for the proximal tubules, loops of Henle, collecting ducts or blood vessels. Furthermore, no noticeable immunosignals could be detected in lung, heart, thymus, spleen, lymph node or liver tissue.
To examine FAM159B expression in the pancreatic islets in more detail, we performed additional double-labelling fluorescence experiments for glucagon and insulin, which confirmed the presence of FAM159B in insulin-producing β-cells and, to some extent, in glucagon-releasing α-cells ( Figure 4). A distinct staining was detected in the pyramidal cells of the human cortex, tri geminal ganglia, dorsal root and intestinal ganglia, pancreatic islets, endocrine cells o the bronchopulmonary and intestinal tract and syncytiotrophoblasts of the placenta ( Figure 3). In addition, slight immunostaining was noticed in bronchial and intestina epithelia, in the epithelium of the larger bile ducts in the liver and in that of th gallbladder. Within human kidneys, the adaptor protein was found in mesangial cells o the glomeruli, the visceral and parietal layer of Bowman's capsule and the distal tubule ( Figure 3E). No expression of FAM159B was observed for the proximal tubules, loops o Henle, collecting ducts or blood vessels. Furthermore, no noticeable immunosignal could be detected in lung, heart, thymus, spleen, lymph node or liver tissue.
To examine FAM159B expression in the pancreatic islets in more detail, we per formed additional double-labelling fluorescence experiments for glucagon and insulin which confirmed the presence of FAM159B in insulin-producing β-cells and, to some extent, in glucagon-releasing α-cells ( Figure 4).  . Double-labelling immunofluorescence analysis of human pancreatic islets. Sections were dewaxed and microwaved in citric acid. Adjacent tissue sections were incubated with rabbit polyclonal anti-FAM159B antibody HPA011778 together with mouse monoclonal antibodies against insulin or glucagon. Labelling for FAM159B was visualised using a Cy3-conjugated anti-rabbit antibody (red) and for insulin or glucagon using an Alexa-Fluor-488-conjugated anti-mouse antibody (green). Scale bar: 100 µm.
Fluorescent staining of the pancreas for FAM159B additionally revealed membranous expression in the acinar cells of the exocrine pancreas ( Figure 5), which was not evident when the samples were evaluated with light microscopy. . Double-labelling immunofluorescence analysis of human pancreatic islets. Sections were dewaxed and microwaved in citric acid. Adjacent tissue sections were incubated with rabbit polyclonal anti-FAM159B antibody HPA011778 together with mouse monoclonal antibodies against insulin or glucagon. Labelling for FAM159B was visualised using a Cy3-conjugated anti-rabbit antibody (red) and for insulin or glucagon using an Alexa-Fluor-488-conjugated anti-mouse antibody (green). Scale bar: 100 µm.  . Double-labelling immunofluorescence analysis of human pancreatic islets. Sections were dewaxed and microwaved in citric acid. Adjacent tissue sections were incubated with rabbit polyclonal anti-FAM159B antibody HPA011778 together with mouse monoclonal antibodies against insulin or glucagon. Labelling for FAM159B was visualised using a Cy3-conjugated anti-rabbit antibody (red) and for insulin or glucagon using an Alexa-Fluor-488-conjugated anti-mouse antibody (green). Scale bar: 100 µm.
Fluorescent staining of the pancreas for FAM159B additionally revealed membranous expression in the acinar cells of the exocrine pancreas ( Figure 5), which was not evident when the samples were evaluated with light microscopy. Figure 5. Immunofluorescence analysis of human exocrine pancreas. Sections were dewaxed and microwaved in citric acid. Tissue sections were incubated with rabbit polyclonal anti-FAM159B antibody HPA011778, followed by Cy3-conjugated anti-rabbit antibody (red). Scale bar: 100 µm. Figure 5. Immunofluorescence analysis of human exocrine pancreas. Sections were dewaxed and microwaved in citric acid. Tissue sections were incubated with rabbit polyclonal anti-FAM159B antibody HPA011778, followed by Cy3-conjugated anti-rabbit antibody (red). Scale bar: 100 µm.

Immunohistochemical Detection of FAM159B Expression in Different Human Tumours and Tumour Cell Lines
The distribution pattern of FAM159B in the different human tumour samples investigated is summarised in Table 1, while Figure 6 shows representative results of these immunostaining experiments.  Again, both cytoplasmic and membranous staining patterns were observed. Furthermore, considerable inter-and intra-individual variability in FAM159B expression was noticed. For several samples, only very small sections of the tumour showed a strong staining signal, while the remainder lacked any expression, resulting in an overall low immunoreactivity score (IRS) value. Overall, moderate-to-strong FAM159B expression (average IRS > 5) was seen in pituitary adenomas, medullary and anaplastic thyroid carcinomas, parathyroid adenomas, adenocarcinomas of the lung, SCLCs, ovarian carcinomas and lymphomas. Low expression of FAM159B (3 ≤ IRS ≤ 5) occurred in papillary thyroid carcinomas, squamous cell carcinomas of the lung, gastric adenocarcinomas, colon carcinomas, pancreatic adenocarcinomas, breast carcinomas, cervical cancer and melanomas. No or very weak expression (IRS < 3) was found in glioblastomas, follicular thyroid carcinomas, gastrointestinal stromal tumours, hepatocellular and cholangiocellular carcinomas, renal clear cell cancer, urinary bladder cancer, pheochromocytomas, endometrial cancer, prostate and testicular cancer and sarcomas (Table 1).
With  Again, both cytoplasmic and membranous staining patterns were observed. Furthermore, considerable inter-and intra-individual variability in FAM159B expression was noticed. For several samples, only very small sections of the tumour showed a strong staining signal, while the remainder lacked any expression, resulting in an overall low immunoreactivity score (IRS) value. Overall, moderate-to-strong FAM159B expression (average IRS > 5) was seen in pituitary adenomas, medullary and anaplastic thyroid carcinomas, parathyroid adenomas, adenocarcinomas of the lung, SCLCs, ovarian carcinomas and lymphomas. Low expression of FAM159B (3 ≤ IRS ≤ 5) occurred in papillary thyroid carcinomas, squamous cell carcinomas of the lung, gastric adenocarcinomas, colon carcinomas, pancreatic adenocarcinomas, breast carcinomas, cervical cancer and melanomas. No or very weak expression (IRS < 3) was found in glioblastomas, follicular thyroid carcinomas, gastrointestinal stromal tumours, hepatocellular and cholangiocellular carcinomas, renal clear cell cancer, urinary bladder cancer, pheochromocytomas, endometrial cancer, prostate and testicular cancer and sarcomas (Table 1).
The median follow-up time was 59.7 months overall. One hundred and fifty-eight (56.8%) patients were alive at the end of the follow-up period, 87 (31.3%) had died due to tumour-related causes and 33 (11.9%) had no data available. The median survival time of patients who died during follow-up was 27.7 months, differing significantly by sex (25 months for males vs. 43.8 months for females).

FAM159B Expression Pattern
Representative stainings of different BP-NEN and GEP-NEN are shown in Figure 7. Again, both cytoplasmic and membranous expression was observed, and substantial variation in immunostaining across individual patients and sometimes even between different samples originating from the same patient was noticed. These findings are illustrated by the lengths of the boxes and whiskers in Figure 8b. For pancreatic tumours, for example, the IRS values ranged from 0 (no expression) to 12 (maximum expression). Regarding the percentage of FAM159B-positive tumours (IRS ≥ 3) (Figure 8a) and the extent of expression (Figure 8b), the protein was most strongly expressed in SCLC (100%; median IRS: 7.5), followed by neuroendocrine tumours from the gut (88.2%; median IRS: 6.5), colon (81.8%; median IRS: 6.0), rectum (86.7%; median IRS: 7.0) and pancreas (83.3%; median IRS: 6.1), as well as in TC (86.4%; median IRS: 6.1). Of all GEP-NEN tested, tumours located in the ileum showed the lowest FAM159B expression (46.6%; median IRS: 3).
tumour-related causes and 33 (11.9%) had no data available. The median survival time of patients who died during follow-up was 27.7 months, differing significantly by sex (25 months for males vs. 43.8 months for females).

FAM159B Expression Pattern
Representative stainings of different BP-NEN and GEP-NEN are shown in Figure 7.

Correlations with Clinical Data
Analysing the collective data of all BP-NEN and GEP-NEN revealed a significant difference between the FAM159B IRS scores for patients with or without lymph node metastases at diagnosis. Patients without lymph node metastases showed higher FAM159B IRS values (mean ± SEM: 5.817 ± 0.366 and 4.797 ± 0.280, respectively; Mann-Whitney U test: p = 0.035). In contrast, a significant positive correlation was observed between FAM159B IRS values and tumour grade (r sp = 0.204; p < 0.001) and levels of the proliferation marker Ki-67 (r sp = 0.306; p < 0.001). Nonetheless, Kaplan-Meier analysis revealed that FAM159B expression does not influence patient overall survival. No difference between FAM159B-positive or -negative tumours was observed when using IRS values of 3 (threshold of positivity) or 6 (close to the overall median IRS value of 5.7) as the cut-off value. When analysing interrelationships with several typical markers of, and receptors for, neuroendocrine tumours determined on the same set of samples as part of previous studies [6][7][8], a correlation was found between the IRS of FAM159B and the IRS values of the dopamine receptor D2, somatostatin receptors (SST)1, 3, 4 and 5, chemokine receptor CXCR4 and programmed death ligand 1 (PD-L1) ( Table 2).
If only the BP-NEN were considered, no correlation between the IRS values of FAM159B and the presence of lymph node metastases was seen, although the positive correlation between FAM159B expression with tumour grade (r sp = 0.139; p = 0.004) and Ki-67 index (r sp = 0.218; p < 0.001) persisted. Again, Kaplan-Meier analysis could not demonstrate any statistically significant differences between patients with FAM159B-positive or -negative tumours. However, a positive correlation between IRS values of FAM159B and those of SST4 and CXCR4 was noted, as well as a trending positive association with PD-L1 (Table 2).    Analyses of only GEP-NEN revealed significantly higher FAM159B IRS values in patients free of lymph node metastases vs. those with metastases (mean ± SEM: 5.979 ± 0.624 vs. 4.431 ± 0.296, respectively; Mann-Whitney U test: p = 0.041). As previously observed, FAM159B IRS was significantly positively correlated with tumour grade (r sp = 0.198; p < 0.001) and Ki-67 levels (r sp = 0.279; p < 0.001). Similarly, Kaplan-Meier analysis showed no influence of FAM159B expression on overall survival of patients. When evaluating the associations between FAM159B and typical neuroendocrine tumour markers, positive associations were observed between the IRS values of FAM15B and those of the dopamine receptor D2, SST1, SST2, SST3, SST5, CXCR4 and PD-L1.
Finally, because double-labelling experiments have shown the presence of FAM159B in insulin-producing β-cells and in some glucagon-producing α-cells, pancreatic neuroendocrine tumours were additionally evaluated for insulin or glucagon expression. Here, a significant relationship was found between the IRS values of FAM159B and those of insulin (r sp = 0.236; p = 0.016) but not of glucagon (r sp = 0.162; p = 0.096). Furthermore, a positive correlation was observed between the IRS values of FAM159B and neuron-specific enolase (NSE) (r sp = 0.217, p = 0.035) but not chromogranin A (r sp = −0.029; p = 0.785).

Western Blot Analysis
The specificity of HPA011778 was demonstrated in BON-1 cells that endogenously express FAM159B using Western blot analysis. The antibody selectively detected the adaptor protein in the supernatant (cytoplasmic fraction) of prepared cell lysates. As no signal was detected when analysing the WGA bead fraction, FAM159B seems to lack noticeable glycosylation within BON-1 cells. The specificity of the antibody for FAM159B was further demonstrated by transfection of BON-1 cells with a specific siRNA and by use of pre-adsorption controls with the immunising peptide. We also confirmed the adaptor protein's previously predicted size of approximately 17 kDa both in BON-1 cells and in mouse pancreas.

Immunocytochemistry
We applied immunocytochemistry to characterise FAM159B subcellular localisation. Endogenously expressed FAM159B in BON-1 cells is found within the cytosol, most likely within distinct vesicles or associated with other proteins. The FAM159B immunosignal was significantly diminished after BON-1 cells were transfected with a specific siRNA. The reduction in signal intensity represents successful transfection and thus decreased protein synthesis. Because the cells were incubated for 24 h after the start of transfection, FAM159B seems to have a lifespan of approximately 24 h, as the signal intensity would not be reduced if the lifespan was much longer. No signal would have been detected at this time point if FAM159B's lifespan was much shorter than 24 h.

Immunohistochemistry
In formalin-fixed, paraffin-embedded human tissue samples, the polyclonal anti-FAM159B antibody yielded a highly efficient and selective immunohistochemical staining of distinct cell populations. Through positive staining of pancreatic islets and endocrine cells of the gastrointestinal tract, the only existing immunohistochemical data of FAM159B expression could be confirmed [2,4]. As another proof of specificity, the immunosignals were completely abolished after pre-absorption of the antibody with its immunising peptide.

FAM159B Expression in Normal Human Tissues
In the present study, for the first time, FAM159B expression has been evaluated and visualised in various normal human tissues using immunohistochemistry and doublelabelling experiments. A strong expression of FAM159B was found in particular in neuronal structures such as pyramidal cells of the cortex, the trigeminal ganglia, dorsal root and enteric ganglia and neuroendocrine tissues and cells, including pancreatic islets and neuroendocrine cells of the bronchopulmonary and gastrointestinal tracts, but also in syncytiotrophoblasts of the placenta. Because all these tissues and cells are involved in the release of various neurotransmitters and hormones, FAM159B may directly or indirectly play a role in their secretory function. In addition to neuronal and neuroendocrine cells, FAM159B was found to be expressed in various epithelia, such as bronchial, gastrointestinal, bile duct and gallbladder epithelia and in epithelia of the distal tubules of the kidney. At these sites, FAM159B may be involved in ion transport processes. The presence of FAM159B in neuroendocrine cells of the intestinal tract and in pancreatic islets has been shown previously [2][3][4] and has been demonstrated to be associated with exocytosis and with Ca 2+ and Na + ion currents [3]. However, the literature disagrees regarding which cells of the pancreatic islets express the protein. Whereas Danielsson et al. [2] proposed FAM159B as a potential marker for total islet mass, Camunas-Soler et al. [3] concluded from their studies that FAM159B is solely a marker of β-cell subsets. In our investigation, the adaptor protein showed homogenous expression throughout the whole of the islets under light microscopy. Subsequent immunofluorescence double-labelling experiments, however, revealed FAM159B to be clearly present in insulin but only to some extent in glucagon-producing islet cells. The assumption of a predominant presence of FAM159B in insulin-secreting cells is further corroborated by a tendency towards positive correlation between expression of FAM159B and insulin, but not glucagon, in pancreatic neuroendocrine tumours.

FAM159B Expression in Human Neoplastic Tissues
In the present study, a wide range of different human tumour entities and cancer cell lines was evaluated for possible expression of FAM159B. To date, no respective data are available from the literature. Our immunohistochemical studies revealed strong expression of FAM159B in tumours of endocrine or neuroendocrine origin, such as pituitary adenomas, BP-NEN and GEP-NEN, medullary and anaplastic thyroid carcinomas and parathyroid adenomas. This finding may be due to the fact that moderate-to-strong expression of FAM159B is normally observed in the corresponding non-neoplastic endocrine and neuroendocrine cells. However, intense FAM159B expression was not confined to tumours of endocrine or neuroendocrine origin. A moderate-to-strong presence of the protein was also found, e.g., in lung adenocarcinomas, ovarian carcinomas and lymphomas. Additionally, in many other tumour entities (despite overall low IRS values), individual tumours showed high FAM159B expression. In contrast, pheochromocytomas, neuroendocrine tumours originating from the adrenal medulla, did not noticeably express the adaptor protein. Fittingly, cancer cell lines with or without a neuronal or neuroendocrine background showed similarly strong immunosignals. Additionally, in BP-NEN and GEP-NEN, distinct differences in FAM159B expression were observed depending on the origin of the primary tumour. Together, these findings suggest functions of FAM159B that go beyond direct or indirect participation in neurotransmission and/or hormone secretion. Correlations with different markers for neuroendocrine tumours revealed a positive relationship between FAM159B and NSE (but not with chromogranin A), as well as with various receptors and membrane proteins commonly expressed in these tumours (but also in others), associated either with good prognosis, such as the D2 dopamine receptor and the different somatostatin receptors or with a negative prognosis, such as the chemokine receptor CXCR4 and PD-L1 [7,9]. Therefore, it is not surprising that no association between FAM159B expression and patient outcomes was seen. This finding may also explain the contradictory results of a positive association between FAM159B expression and tumour proliferation rate or grade and higher FAM159B levels in patients without lymph node metastases.

Antibody
The rabbit polyclonal anti-FAM159B antibody (HPA011778) was purchased from Atlas Antibodies (Bromma, Sweden). The sequence of the peptide used for the immunisations of the rabbits was as follows: TKPQRLDTGLKLQHLEASSTQEGKSNGKTKALNSNAAS-NATN ETYYEADDIIQEKTMDATQIHIA. The respective peptide PrEST Antigen FAM159B (APrEST71583) was also obtained from Atlas Antibodies (Bromma, Sweden).

Western Blot Analysis
BON-1 cells (DMSZ, Braunschweig, Germany), a neuroendocrine tumour cell line endogenously expressing FAM159B, were seeded into 60 mm Petri dishes and grown to 80% confluence. Cells were lysed in detergent buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, 100 mM phenylmethylsulfonylfluoride, 10 mg/mL leupeptin, 5 mg/mL aprotinin, 1 mg/mL pepstatin A). For detection of FAM159B in mouse pancreas, 50 mg of tissue was weighed out, 200 µL of the detergent buffer was added and the pancreas was sonicated for 10 s. Afterwards, the samples were gently inverted for 1 h at 4 • C before centrifugation for 30 min at 14,800 g at 4 • C. All samples were then treated with wheat germ lectin-agarose (WGA) beads. The supernatant of the samples, as well as the bead fractions, were subjected to 10% SDS-polyacrylamide gel electrophoresis and immunoblotted onto polyvinylidene fluoride membranes. The blots were then incubated with rabbit polyclonal anti-FAM159B antibody HPA011778 at a dilution of 1:500 overnight at 4 • C, followed by incubation with a peroxidase-conjugated secondary anti-rabbit antibody (dilution 1:5000; Santa Cruz Biotechnology, Dallas, TX, USA) and enhanced chemiluminescence detection (Amersham, Braunschweig, Germany).
When indicated, the expression of endogenous FAM159B in BON-1 cells was silenced using a chemically synthesised, single-stranded RNA oligonucleotide (siRNA; ID 264018; Ambion by Life Technologies (Thermo Fisher Scientific), Waltham, MA, USA) according to the manufacturer's instructions. An empty vector was used as a negative control (Santa Cruz Biotechnology, Dallas, TX, USA).
For adsorption controls, HPA011778 was pre-incubated for 2 h at room temperature with 10 µg/mL of the peptide PrEST antigen (Atlas Antibodies, Bromma, Sweden) used for rabbit immunisations.

Immuncytochemistry
BON-1 cells were grown overnight on glass coverslips until 80% confluence was achieved. After washing of the cells with phosphate-buffered saline (PBS), cells were fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate buffer (pH 6.9) for 20 min at room temperature. After thorough washing with PBS, cells were incubated with anti-FAM159B HPA011778 overnight at 4 • C, followed by incubation with an Alexa-Fluor-488-conjugated secondary antibody (Invitrogen, Karlsruhe, Germany; dilution 1:5000) for 2 h at room temperature. Samples were then mounted (Invitrogen Fluoromount-G, with DAPI; Thermo Fisher Scientific, Waltham, MA, USA) and examined using a Zeiss LSM 510 META laser scanning confocal microscope (Jena, Germany).
If required, endogenous FAM159B expression in BON-1 cells was silenced using a chemically synthesised, single-stranded RNA oligonucleotide (siRNA; ID 264018; Ambion by Life Technologies (Thermo Fisher Scientific), Waltham, MA, USA) according to the manufacturer's instructions. An empty vector was used as a negative control (Santa Cruz Biotechnology, Dallas, TX, USA).
When indicated, HPA011778 was pre-adsorbed for 2 h at room temperature with 10 µg/mL of the peptide PrEST antigen (Atlas Antibodies, Bromma, Sweden) used for rabbit immunisations.

Tumour Specimens
For the initial assessment of FAM159B expression in various human tumour specimens, 259 formalin-fixed and paraffin-embedded tumour samples (Table 1) were obtained from the Department of Pathology of the Ernst Moritz Arndt University (Greifswald, Germany) and the Laboratory of Pathology and Cytology Bad Berka (Bad Berka, Germany). Several tumour specimens contained neighbouring non-malignant tissue that was also analysed. Furthermore, tumour-free human tissue samples from the cortex, lung, heart, liver, stomach, gut, pancreas, kidney, spleen, thymus, lymph nodes and placenta (n = 2-5 each site) were also evaluated. These specimens were obtained from the Department of Pathology of the Ernst Moritz Arndt University (Greifswald, Germany) and the Laboratory of Pathology and Cytology Bad Berka (Bad Berka, Germany).

Statistics
SPSS 25.0.0.0 (IBM, Armonk, NY, USA) was used for the statistical analyses. Because the data were not normally distributed, a Kolmogorov-Smirnov test, Kruskal-Wallis test, Mann-Whitney test, chi-square test, Kendall's τ-b test or Spearman's rank correlation was performed. For survival analysis, the Kaplan-Meier method with a log-rank test was used. P values ≤ 0.05 were considered statistically significant.

Conclusions
We thoroughly characterised the polyclonal rabbit anti-human FAM159B antibody HPA011778 and demonstrated its suitability for both Western blot and immunocytochemistry analyses in basic research, as well as for visualising FAM159B expression in formalinfixed, paraffin-embedded tissue samples. By applying this antibody, we could detect FAM159B expression in various non-neoplastic and neoplastic tissues and cancer cell lines. Our results also implicate FAM159B in the direct or indirect regulation of diverse membrane proteins, including receptors, ion channels or others such as PD-L1, that may be typically (but not exclusively) expressed in neuronal and (neuro)endocrine tissues. In this context, FAM159B may also regulate secretory functions, among other actions. Informed Consent Statement: For this type of study, formal consent was not required. All data were recorded and analysed anonymously.

Data Availability Statement:
The data that support the findings of this study are all contained within the article.
Conflicts of Interest: D.K. received funding and support for travel to meetings from IPSEN and PFIZER. All other authors declare no competing interests.